Traditional cell culture is usually carried out in a Petri dish, and most commonly used cell biological techniques rely on the use of cultured cells on 2D platforms. As is known, the mechanical properties of cell substrates affect cell differentiation, growth and motility. Thus, the mechanical properties of a material are important to consider when designing a cell scaffold. To mimic an actual cell environment, it is imperative to create 3D functional tissue models using 3D biomanufacturing methods rather than 2D patterning ones. In addition, the manufacturing process and/or materials must be cell-friendly. Current fabrication systems for biological scaffolds utilize approaches such as bio-inkjet printing and raster laser projection, which are limited in scalability and speed, and cannot create complex structures and scalable tissue construct. Cast molding approaches can achieve basic geometries but cannot reproduce complex 3D geometries with intricate biomimetic features and aspect ratios. Laser micro-stereolithography (laser-μSL) techniques have grown to be popular for their abilities to create 3D scaffolds [Mapili et al., 2005; Gansel et al., 2009]. However, scaffold fabrication using laser-μSL is usually slow due to point-by-point laser scanning Many existing layer-by-layer stereolithographic methods do not feature a dynamically changing mask and simultaneous stage movement and therefore cause undesired layer artifacts that may alter cellular responses and not reflect the true native physiology.
One approach that has been commercialized by Carbon3D, Inc. (Redwood City, Calif.) for immersion printing of polymer nanostructures involves projecting an image through the transparent floor of a liquid resin reservoir while gradually lifting an immersed substrate away from the floor as the resin is cured on the bottom of the substrate. The oxygen permeable transparent floor of the bath allows for a “dead zone” of dissolved oxygen to inhibit polymerization at the floor of the bath. The techniques used in the Carbon3D system are described in numerous U.S. patents, issued and pending, to Joseph M. DeSimone, et al., including U.S. Pat. No. 8,263,129, which is incorporated herein by reference. The reservoir and substrate of the Carbon3D system remain stationary, with only the immersed substrate moving within the z-axis to lift the printed object away from the floor as printing advances, which limits the varieties of shapes that can be created. Further, the technology's reliance on immersion limits the types and combinations of materials that may be used.
Conventional stereolithographic approaches are not well suited for high-throughput fabrication of complex cell-supportive 3D microstructures, particularly within substrates such as multi-well plates that are commonly used in the life sciences. These shortcomings severely limit widespread adoption of 3D printed cell culture methods since researchers often rely on products configured to interface with commonly used lab instruments integral to established experimental workflows. Multi-well cell culture plates (in addition to microscope slides, Petri dishes, cell culture flasks, etc.) often serve as the de facto standard upon which specialized culture environments are designed. Many 3D hydrogel cell culture platforms feature biocompatible and/or biologically-derived materials that have been polymerized or otherwise manufactured, albeit typically as unpatterned bulk structures, within multi-well plates. Similarly, high throughput cell culture systems with integrated microfluidics or multi-electrode arrays have also been developed on multi-well or standard microscope slide formats.
Commercial 3D bioprinters capable of printing directly into multi-well plates are available from a number of companies, including the BioAssemblyBot™ bioprinter from Advanced Solutions, Inc. (Louisville, Ky., US), the Regenova® bioprinter from Cyfuse Biomedical K.K. (Tokyo, JP), and the NovoGen MMX™ bioprinter from Invetech, Inc. (Melbourne, AU). These systems use a raster-scanning approach (i.e., “inkjet-like”) to fabricate the 3D constructs via extrusion of the biomaterial and, thus, suffer from inherent limitations in scalability, resolution, and material selection.
Highly-specified 3D cell culture microenvironments can be utilized in a broad range of physiological contexts, including in vitro neuronal cultures. The goal of studying isolated neural cultures is to examine and probe a simple in vitro system that can represent physiologically relevant models. Isolated neural cultures are a cornerstone of neuroscience research, yet their utility in reflecting native physiology is limited due to their inherently indeterministic connectivity. Although a large sum of neurophysiological data that details the function of monolayers of neurons is widely available, the conditions used for these cultures vary drastically from the ones present in native tissue. Thus, it is reasonable to assume that the behavior of 2D cultures is not a good representation of the complex system that is the in vivo neural physiology.
Recent work demonstrates that growing neurons in 3D scaffolds with incorporated glia provides drastic morphological and electrophysiological differences in comparison to neural networks grown in 2D cultures. This is attributed to the fact that 3D neural scaffolds better resemble the complex neural environment present in vivo.
In vitro replication of neural circuitry may help elucidate essential parts of the neural milieu and facilitate testing of connectivity models that contribute to higher-level processes. Additionally, as patient-specific induced pluripotent stem cells (iPSCs) are adopted for in vitro disease models, controlling the functional arrangement of neural populations may be critical to recreate normal versus pathological neural circuits. Conventional means for neuronal patterning are often restricted to a 2D context and use substrates that do not reflect native mechanochemical properties. On the other hand, methods to generate soft 3D scaffolds can be costly, time-consuming, or limited to simple geometric features. Given the limitations of current platforms, there is a need to establish a high-throughput means for deterministically controlling and systematically investigating the network dynamics of fundamental neural circuits in more physiologically representative, soft 3D environments.
Engineering a simplified neural circuit with the flexibility for systematically increased complexity provides an attractive model for studying neural wiring and functional connectivity. Current high-density recording and stimulation techniques via surface microelectrode arrays in vitro provide a wealth of information on the state of the network, and the utility of an in vitro model extends also to drug screening given its high throughput nature. Recent innovations in culturing neurons and guiding neural growth offer some control over network connectivity, cell density, and neural phenotypes to achieve some order and simplicity in the network. Notably, the advent of 3D cultures where neurons are grown within hydrogel scaffolds (with thicknesses of at least 10 cell diameters) have served to address the limitations of the 2D model by: 1) maintaining more relevant cell-cell/cell-matrix interactions, 2) protecting neurons from pH changes in culture media, 3) employing a mechanically softer interface that reflects native physiology more so than hard substrates in 2D cultures, and 4) providing high surface area for growth and migration. Thus, 3D constructs are essential to progress in the study of isolated neural networks and the development of physiologically relevant “brain-on-a-chip” systems. Recent advances in this area demonstrate the potential for sustaining neurons in 3D scaffolds for weeks while guiding neural growth in controlled geometric patterns. However, challenges remain in: 1) achieving high spatial resolution and density in recording and stimulation, 2) simplifying costly and laborious fabrication techniques, and 3) producing heterogeneous co-cultures of neurons and glia, which has proven critical for the proper function of neurons.